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Lyonisation of the X Chromosome
769
LEADING ARTICLES
XXX, and XXXX would present all exactly the same clinical picture, as would XY, XXY, XXXY, and
THE LONDON
LANCET 12
OCTOBER
1963
X Chromosome MARY LYON proposed her hypothesis on the behaviour of X chromosomes in XX females in 1961, originally to explain her findings on the coat colour of mice carrying sex-linked colour genes.1 A colloquium on chromosome anomalies and D.N.A., held in Oporto on Sept. 12-14, devoted a good part of its time to this subject.
Lyonisation of the
general, the hypothesis seems to be now widely accepted as the best available explanation of the known facts in mammals. In the male the single X remains In
extended and active. In the female one X is extended and active, the other remains aggregated (hence the visible sex-chromatin), synthesises its D.N.A. later than all the active chromosomes, and takes little or no active part in directing cellular activities. LYON postulated that the choice as to which of two X chromosomes is to be inactivated has to be made at a definite point of foetal development, that it is a choice made at random and independently by each cell present in the foetus at that time, and that once made it is irrevocable and transmitted to all the descendants of the cell. This " Lyonisation " (a convenient word invented at the colloquium) occurs in man presumably about the 12th day: and all the cells in the body of a woman can be divided into two classes-those with an active maternal X chromosome and those with an active paternal, according to what happened to their ancestor cell at the time of Lyonisation. The eye of a woman heterozygous for such an X-carried gene as colourblindness will thus contain clones of colourblind rods alternating with normal clones, and similarly if she is heterozygous for glucose-6-phosphatedehydrogenase deficiency her red cells will be a mixture of deficient and normal cells.2 Only one important piece of discordant evidence has emerged recently. Women heterozygous for the sexlinked blood-group Xga should have a mixture of Xg(a+) and Xg(a-) red cells. In fact, as was demonstrated elegantly and conclusively by a fluorescentantibody technique, all their red cells are Xg(a&plusdot;).3 But this is not fatal: a segment of the Lyonised X chromosome may remain active; the antigen may be diffusible; or (unlikely in this case, but very likely in some other instances) one race of cells may have a selective advantage over the other. Obviously, however, the general picture of the human sex-chromosome anomalies is difficult to reconcile with the total inactivation of all X chromosomes beyond the first assumed by the hypothesis in its extreme form. If Lyonisation of all Xs beyond the first was total, the number of Xs would make no difference; and XO, XX, 1. 2. 3.
Lyon, M. F. Nature, Lond. 1961, 190, 372. See Lancet, 1962, ii, 29. Beutler, E., Yek, M., Fairbanks, V. F. Proc. Nat. Acad. Sci. 1962, 48, 9. Reed, T. E., Simpson, N. E., Chown, B. Lancet, Aug. 31, 1963, p. 467.
’XXXXY. The loss of an X in Turner’s syndrome, or its gain in XXX or in XXY Klinefelter’s syndrome, obviously does make a difference. But it must be admitted that no hypothesis yet presented can wholly be reconciled with the facts. If one assumes that a small segment of the X is active despite Lyonisation (as the in each sex taken Xga findings suggest) the separately fit well enough: for instance, the extra active segment in XXX and XXY could produce the same effect as trisomy of a very small chromosome, which is not unreasonable. When one compares the sexes, however, the discrepancies are obvious. One can pair off Turner’s with a normal male, XXY with a normal female, and XXX with XXXY, each of these should have the same total X activity. Yet in none of these pairs do the partners have the same degree of congenital defect. For the lowered intelligence in XXY Klinefelter’s syndrome one cannot blame the active X or the Y, which are present in a normal male, or the second inactivated X, which is present in a normal female; nor can one blame the mere presence of three chromosomes, for XYY is not associated with mental defect.
findings
alternative line of reasoning there must be a small paired segment on both X and Y chromosomes. This is particularly satisfactory in explaining Turner’s syndrome, for where both normal XX and normal XY would have two paired segments each, XO would have only one. The hypothesis limps rather with XXX and XXY: both of these would have three paired segments and so effectively be trisomic, which fits well enough with their general character; but they should be identical trisomies on every score except sexual differentiation, and this they are not (the incidence of mental defect is much higher in XXX, for one thing). It falls down altogether over XYY, which ought to be trisomic for the paired segment but is a normal male so far as is known. If one could forget about XYY (and it is just possible that identification of the extra chromosome as Y in some cases is false) it is possible to synthesise the two hypotheses just considered, and perhaps achieve something nearer the truth by assuming that both active segments of the inactive X and paired segments are concerned, their effects being summed: but we are clearly far from a wholly satisfactory answer. It seems, incidentally, that the cytogeneticists cannot yet decide certainly whether or not the X and the Y, which pair off end-to-end rather than as usual side by side, have in fact any true paired segment in common. Matters are a little simplified if we can dissociate the gonadal defects of the sex-chromosome disorders from the somatic disorders, and this can be done on a relatively simple hypothesis. It is necessary to assume only that the differentiation of the gonad depends simply on the presence or absence of the Y, but that of the germ cell is independent of this and depends on the presence or absence of a second X. In a normal male the Y produces the testis and absence of the second X produces the spermatogonia. In a normal female the absence of the
By
an
770
Antibodies
produces an ovary, and the second X produces oogonia. In XO Turner’s, however, the absence of Y produces an ovary, and the absence of a second X produces spermatogonia: the two are incompatible and the germ cells die. Similarly XXY Klinefelter’s produces a testis with oogonia, and again most or all of the incompatible germ cells die. An attractive hypothesis, and as far as we know original, but unprovable. Extension to species other than man would have to explain the fertility
Y
of XO mice, but it appears that the situation in these mice may be more complex than originally appeared. Turner’s syndrome remains on many counts the most difficult of the sex-chromosome anomalies to account for. Some of the difficulties have been resolved by the demonstration that mosaicism is common: in particular the varying possibilities of XX/XO and XY/XO mosaicism account for many atypical cases. But classical XO Turner’s syndrome remains as much a problem as ever. The hypothesis just mentioned may account for the gonadal lesion and its consequences, but the relation of the gonad lesion to the other congenital anomaliesdwarfing, webbed neck, coarctation, and the like-has never been clear. A most ingenious hypothesis recently stated in brief by GARTLER and SPARKES4 of Seattle offers a
possible explanation.
GARTLER and SPARKES suppose that at the time of Lyonisation the single X chromosome of the XO cell can either become inactivated or not, as a matter of chance. If it is not inactivated, the result is to all intents and purposes a normal cell with one active X. If it is inactivated, the cell dies. The result is that about half the cells of the foetus die all at once, but the cells that remain behave as if they were normal. It is, of course, a matter of pure speculation whether such a loss of a randomly chosen half of the cells of the foetus on about the 12th day would produce the stunting and other defects of Turner’s syndrome; but, on the face of it, this seems not at all improbable. If correct, this would account for the profound difference-notably in the lack of mental defect - between Turner’s syndrome and the trisomies, all of which have a kind of family resemblance: the defects in one would depend on a single catastrophe leaving behind normal cells, whereas in the others the defects depend on the continuing presence of abnormal cells. The hypothesis fits particularly well with the facts of the usual form of isochromosome Turner’s syndrome, and indeed seems to have been framed originally to meet some difficulties in the unmodified application of Lyon’s hypothesis to these cases.5The Oporto colloquium thought there might be difficulty in its application to some other related anomalies (e.g., short-arm X isochromosomes), but more details of the cases concerned are needed. On the whole the idea seems a good one. ,
This brief review of the pathogenesis of the sexchromosome anomalies shows that we are still very far’ from total understanding. But it seems extremely likely that this curious behaviour of the X uncovered by LYONr will prove an essential part of the process. ’
’
4. Gartler, S. M., Sparkes, R. S. ibid. Aug. 24, 5. Muldal, S., Gilbert, C. W., Lajtha, L. G., Fraccaro, M. ibid. 1963, i, 861.
issue of the British Medical Bulletin devoted to antibodies, A. A. MILES reflects that nearly all the immunological phenomena which occupy our attention today were uncovered in the thirty years that succeeded VON BEHRING and KITASATo’s discovery in 1890 of the existence of antitoxic activity in certain immune sera. Detailed knowledge of the nature of antibody dates from less than twenty-five years ago when TISELius and KABAT identified antibodies as y-globulins. From this discovery, immunochemical knowledge has developed steadily to the point of recognising that the fraction of the serum proteins which encompasses antibody activity is in fact a complex and heterogeneous mixture whose components-the immunoglobulins-are divisible for practical purposes into different types on the basis of electrophoretic, ultracentrifugal, and chromatographic behaviour. To this knowledge of the character of y-globulin is being added an analysis of the immunological properties of its molecules as antigens; and the recent elucidation of their chemical structure in terms of constituent fragments and polypeptide chains has, already, led to important advances relating these components to the behaviour and function of the whole antibody molecule. The structural basis of biological activity and specificity of antibody is a meeting-point for many different lines of current immunological inquiry. Immune tolerance, rejection of tissue grafts, and the immunopathology of allergy and autoimmunity all INTRODUCING
1963, p. 411. Lindsten, J., Rowley, J.
an
involve
problems relating more or less closely to these properties of circulating antibody. The nature of the antigen-antibody reaction at a molecular level is basically important. It seems to be a matter of complementary configuration rather than chemical combination, its specificity arising from the close intermolecular fit necessary to allow the operation of the very short-range forces which bind the reactants together. The mass action association constant for any particular antigenantibody reaction yields thermodynamic data about, for example, free-energy exchange which indicate the of such bonds. Measurements made in several different systems show differences in the values for the equilibrium constant as between protein antigens and haptens, and agree in attributing a valency of approxi-
nature
mately
2
to
antibody.
Equilibrium relationships
between bound and free antibody can be used in a redcell antibody system, as N. C. HUGHES-JONES shows, to determine the number of antigenic sites on an erythrocyte-in the case of the human D Rh antigen, 24,000. Such data also reveal graphically the heterogeneity of affinity for antigen sites which antibody molecules
display. Laboratory versatility in the detection of antigenantibody reactions is continually increasing, and with it the need for defining sensitivities of different methods in terms of minimum amounts of antibody N detected. J. R. MARRACK, in a timely review, provides a yardstick for this purpose. There is commonly discordance 1. Brit. med. Bull.
September,
1963.
LEADING ARTICLES
XXX, and XXXX would present all exactly the same clinical picture, as would XY, XXY, XXXY, and
THE LONDON
LANCET 12
OCTOBER
1963
X Chromosome MARY LYON proposed her hypothesis on the behaviour of X chromosomes in XX females in 1961, originally to explain her findings on the coat colour of mice carrying sex-linked colour genes.1 A colloquium on chromosome anomalies and D.N.A., held in Oporto on Sept. 12-14, devoted a good part of its time to this subject.
Lyonisation of the
general, the hypothesis seems to be now widely accepted as the best available explanation of the known facts in mammals. In the male the single X remains In
extended and active. In the female one X is extended and active, the other remains aggregated (hence the visible sex-chromatin), synthesises its D.N.A. later than all the active chromosomes, and takes little or no active part in directing cellular activities. LYON postulated that the choice as to which of two X chromosomes is to be inactivated has to be made at a definite point of foetal development, that it is a choice made at random and independently by each cell present in the foetus at that time, and that once made it is irrevocable and transmitted to all the descendants of the cell. This " Lyonisation " (a convenient word invented at the colloquium) occurs in man presumably about the 12th day: and all the cells in the body of a woman can be divided into two classes-those with an active maternal X chromosome and those with an active paternal, according to what happened to their ancestor cell at the time of Lyonisation. The eye of a woman heterozygous for such an X-carried gene as colourblindness will thus contain clones of colourblind rods alternating with normal clones, and similarly if she is heterozygous for glucose-6-phosphatedehydrogenase deficiency her red cells will be a mixture of deficient and normal cells.2 Only one important piece of discordant evidence has emerged recently. Women heterozygous for the sexlinked blood-group Xga should have a mixture of Xg(a+) and Xg(a-) red cells. In fact, as was demonstrated elegantly and conclusively by a fluorescentantibody technique, all their red cells are Xg(a&plusdot;).3 But this is not fatal: a segment of the Lyonised X chromosome may remain active; the antigen may be diffusible; or (unlikely in this case, but very likely in some other instances) one race of cells may have a selective advantage over the other. Obviously, however, the general picture of the human sex-chromosome anomalies is difficult to reconcile with the total inactivation of all X chromosomes beyond the first assumed by the hypothesis in its extreme form. If Lyonisation of all Xs beyond the first was total, the number of Xs would make no difference; and XO, XX, 1. 2. 3.
Lyon, M. F. Nature, Lond. 1961, 190, 372. See Lancet, 1962, ii, 29. Beutler, E., Yek, M., Fairbanks, V. F. Proc. Nat. Acad. Sci. 1962, 48, 9. Reed, T. E., Simpson, N. E., Chown, B. Lancet, Aug. 31, 1963, p. 467.
’XXXXY. The loss of an X in Turner’s syndrome, or its gain in XXX or in XXY Klinefelter’s syndrome, obviously does make a difference. But it must be admitted that no hypothesis yet presented can wholly be reconciled with the facts. If one assumes that a small segment of the X is active despite Lyonisation (as the in each sex taken Xga findings suggest) the separately fit well enough: for instance, the extra active segment in XXX and XXY could produce the same effect as trisomy of a very small chromosome, which is not unreasonable. When one compares the sexes, however, the discrepancies are obvious. One can pair off Turner’s with a normal male, XXY with a normal female, and XXX with XXXY, each of these should have the same total X activity. Yet in none of these pairs do the partners have the same degree of congenital defect. For the lowered intelligence in XXY Klinefelter’s syndrome one cannot blame the active X or the Y, which are present in a normal male, or the second inactivated X, which is present in a normal female; nor can one blame the mere presence of three chromosomes, for XYY is not associated with mental defect.
findings
alternative line of reasoning there must be a small paired segment on both X and Y chromosomes. This is particularly satisfactory in explaining Turner’s syndrome, for where both normal XX and normal XY would have two paired segments each, XO would have only one. The hypothesis limps rather with XXX and XXY: both of these would have three paired segments and so effectively be trisomic, which fits well enough with their general character; but they should be identical trisomies on every score except sexual differentiation, and this they are not (the incidence of mental defect is much higher in XXX, for one thing). It falls down altogether over XYY, which ought to be trisomic for the paired segment but is a normal male so far as is known. If one could forget about XYY (and it is just possible that identification of the extra chromosome as Y in some cases is false) it is possible to synthesise the two hypotheses just considered, and perhaps achieve something nearer the truth by assuming that both active segments of the inactive X and paired segments are concerned, their effects being summed: but we are clearly far from a wholly satisfactory answer. It seems, incidentally, that the cytogeneticists cannot yet decide certainly whether or not the X and the Y, which pair off end-to-end rather than as usual side by side, have in fact any true paired segment in common. Matters are a little simplified if we can dissociate the gonadal defects of the sex-chromosome disorders from the somatic disorders, and this can be done on a relatively simple hypothesis. It is necessary to assume only that the differentiation of the gonad depends simply on the presence or absence of the Y, but that of the germ cell is independent of this and depends on the presence or absence of a second X. In a normal male the Y produces the testis and absence of the second X produces the spermatogonia. In a normal female the absence of the
By
an
770
Antibodies
produces an ovary, and the second X produces oogonia. In XO Turner’s, however, the absence of Y produces an ovary, and the absence of a second X produces spermatogonia: the two are incompatible and the germ cells die. Similarly XXY Klinefelter’s produces a testis with oogonia, and again most or all of the incompatible germ cells die. An attractive hypothesis, and as far as we know original, but unprovable. Extension to species other than man would have to explain the fertility
Y
of XO mice, but it appears that the situation in these mice may be more complex than originally appeared. Turner’s syndrome remains on many counts the most difficult of the sex-chromosome anomalies to account for. Some of the difficulties have been resolved by the demonstration that mosaicism is common: in particular the varying possibilities of XX/XO and XY/XO mosaicism account for many atypical cases. But classical XO Turner’s syndrome remains as much a problem as ever. The hypothesis just mentioned may account for the gonadal lesion and its consequences, but the relation of the gonad lesion to the other congenital anomaliesdwarfing, webbed neck, coarctation, and the like-has never been clear. A most ingenious hypothesis recently stated in brief by GARTLER and SPARKES4 of Seattle offers a
possible explanation.
GARTLER and SPARKES suppose that at the time of Lyonisation the single X chromosome of the XO cell can either become inactivated or not, as a matter of chance. If it is not inactivated, the result is to all intents and purposes a normal cell with one active X. If it is inactivated, the cell dies. The result is that about half the cells of the foetus die all at once, but the cells that remain behave as if they were normal. It is, of course, a matter of pure speculation whether such a loss of a randomly chosen half of the cells of the foetus on about the 12th day would produce the stunting and other defects of Turner’s syndrome; but, on the face of it, this seems not at all improbable. If correct, this would account for the profound difference-notably in the lack of mental defect - between Turner’s syndrome and the trisomies, all of which have a kind of family resemblance: the defects in one would depend on a single catastrophe leaving behind normal cells, whereas in the others the defects depend on the continuing presence of abnormal cells. The hypothesis fits particularly well with the facts of the usual form of isochromosome Turner’s syndrome, and indeed seems to have been framed originally to meet some difficulties in the unmodified application of Lyon’s hypothesis to these cases.5The Oporto colloquium thought there might be difficulty in its application to some other related anomalies (e.g., short-arm X isochromosomes), but more details of the cases concerned are needed. On the whole the idea seems a good one. ,
This brief review of the pathogenesis of the sexchromosome anomalies shows that we are still very far’ from total understanding. But it seems extremely likely that this curious behaviour of the X uncovered by LYONr will prove an essential part of the process. ’
’
4. Gartler, S. M., Sparkes, R. S. ibid. Aug. 24, 5. Muldal, S., Gilbert, C. W., Lajtha, L. G., Fraccaro, M. ibid. 1963, i, 861.
issue of the British Medical Bulletin devoted to antibodies, A. A. MILES reflects that nearly all the immunological phenomena which occupy our attention today were uncovered in the thirty years that succeeded VON BEHRING and KITASATo’s discovery in 1890 of the existence of antitoxic activity in certain immune sera. Detailed knowledge of the nature of antibody dates from less than twenty-five years ago when TISELius and KABAT identified antibodies as y-globulins. From this discovery, immunochemical knowledge has developed steadily to the point of recognising that the fraction of the serum proteins which encompasses antibody activity is in fact a complex and heterogeneous mixture whose components-the immunoglobulins-are divisible for practical purposes into different types on the basis of electrophoretic, ultracentrifugal, and chromatographic behaviour. To this knowledge of the character of y-globulin is being added an analysis of the immunological properties of its molecules as antigens; and the recent elucidation of their chemical structure in terms of constituent fragments and polypeptide chains has, already, led to important advances relating these components to the behaviour and function of the whole antibody molecule. The structural basis of biological activity and specificity of antibody is a meeting-point for many different lines of current immunological inquiry. Immune tolerance, rejection of tissue grafts, and the immunopathology of allergy and autoimmunity all INTRODUCING
1963, p. 411. Lindsten, J., Rowley, J.
an
involve
problems relating more or less closely to these properties of circulating antibody. The nature of the antigen-antibody reaction at a molecular level is basically important. It seems to be a matter of complementary configuration rather than chemical combination, its specificity arising from the close intermolecular fit necessary to allow the operation of the very short-range forces which bind the reactants together. The mass action association constant for any particular antigenantibody reaction yields thermodynamic data about, for example, free-energy exchange which indicate the of such bonds. Measurements made in several different systems show differences in the values for the equilibrium constant as between protein antigens and haptens, and agree in attributing a valency of approxi-
nature
mately
2
to
antibody.
Equilibrium relationships
between bound and free antibody can be used in a redcell antibody system, as N. C. HUGHES-JONES shows, to determine the number of antigenic sites on an erythrocyte-in the case of the human D Rh antigen, 24,000. Such data also reveal graphically the heterogeneity of affinity for antigen sites which antibody molecules
display. Laboratory versatility in the detection of antigenantibody reactions is continually increasing, and with it the need for defining sensitivities of different methods in terms of minimum amounts of antibody N detected. J. R. MARRACK, in a timely review, provides a yardstick for this purpose. There is commonly discordance 1. Brit. med. Bull.
September,
1963.